Methods and compositions for preventing or treating calciphylaxis

ABSTRACT

The invention provides methods and compositions for preventing or treating (e.g., slowing the progression of, arresting, and/or reversing) calciphylaxis in a subject in need thereof and, more particularly, the invention relates to methods of using menaquinone-7 (MK-7) and/or menaquinol-7 (MKH2-7) for preventing or treating calciphylaxis in a subject with one or more of the following: diabetes, chronic kidney disease, end stage renal failure, and COPD or a subject undergoing hemodialysis and/or receiving anticoagulant therapy and/or statin therapy.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. ProvisionalPatent Application No. 62/682,794, filed on Jun. 8, 2018, which isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates generally to methods and compositions forpreventing or treating (e.g., slowing the progression of, arresting,and/or reversing) calciphylaxis in a subject in need thereof and, moreparticularly, the invention relates to methods of using menaquinone-7(MK-7) and/or menaquinol-7 (MKH2-7), for preventing or treating (e.g.,slowing the progression of, arresting, and/or reversing) calciphylaxisin a subject with diabetes, chronic kidney disease, end stage renaldisease, or a subject undergoing hemodialysis and/or receivinganticoagulant therapy and/or a subject who has been, or is presently, onstatin therapy and/or suffering from chronic obstructive pulmonarydisorder (COPD).

BACKGROUND

Under normal physiological conditions, plasma calcium and phosphate arepresent at concentrations close to supersaturation levels and, as aresult, may be expected to precipitate in soft tissue (e.g., bloodvessels) as crystalline hydroxyapatite. The observation that thisprocess does not occur in healthy subjects suggested the presence ofpotent chemical and biologic means for blocking pathologic calcification(Price, et al. (2002) “Discovery of a High Molecular Weight Complex ofCalcium, Phosphate, Fetuin, and Matrix-Carboxyglutamic Acid Protein inthe Serum of Etidronate-treated Rats,” JOURNAL BIOL. CHEM. 277 (6):3926-3934).

When the suppression of calcification is disrupted, such as in subjectswith diabetes and chronic kidney disease (CKD), pathologic calcificationof soft tissue (e.g., blood vessels) can occur. It is understood thatdiabetes can lead to CKD and end stage renal disease (ESRD), which ischaracterized by uremia. Uremia can promote the oxidation of Vitamin Khydroquinone (KH2), thereby disrupting the cyclic regeneration ofVitamin K, among other effects. (See, FIG. 1.) In addition, certaintreatments can cause or contribute to Vitamin K dysregulation, includingwarfarin-based anticoagulant therapy and statin therapy. The loss offunctional Vitamin K results in the loss of important regulators ofmineralization, leading to pathologic calcification of tissue. In thecase of arterial calcification, intradermal microvascular thrombosis isobserved to occur, resulting in small vessel blockages and surroundingtissue death.

Calciphylaxis (also referred to as Calcific Uremic Arteriolopathy(CUA)), is a rare, but potentially fatal complication of chronic kidneydisease (CKD), predominantly affecting patients with end-stage renaldisease (ESRD) on hemodialysis. The condition is characterized bycalcification of subcutaneous arterioles and infarctions of the adjacentsubcutis and skin (Janigan et al. (2000) “Calcified subcutaneousarterioles with infarcts of the subcutis and skin (“calciphylaxis”) inchronic renal failure,” AMERICAN JOURNAL OF KIDNEY DISEASES35(4):588-97; Brandenburg et al. (2016) “ERA-EDTA Working Group onCKD-MBD and EUCALNET. Lack of evidence does not justify neglect: how canwe address unmet medical needs in calciphylaxis?” NEPHROL DIALTRANSPLANT. 31(8): 1211-9; Brandenburg et al. (2017) “Calcific uraemicarteriolopathy (calciphylaxis): data from a large nationwide registry,”NEPHROL DIAL TRANSPLANT. 32(1):126-132).

The exact incidence and prevalence of calciphylaxis are unknown(Brandenburg et al. 2017, supra). The estimated annual incidence is <1%and the prevalence is about 4% among chronic hemodialysis patients(Angelis et al. (1997) “Calciphylaxis in patients on hemodialysis: Aprevalence study,” SURGERY 122(6):1083-90; Brandenburg et al. (2014)“Calcific uraemic arteriolopathy: a rare disease with a potentially highimpact on chronic kidney disease-mineral and bone disorder,” PEDIATRNEPHROL. 29(12):2289-98; Brandenburg et al. (2016), supra).

Based on the United States Renal Data System, between 2010 and 2015,there was an increase in the: (i) incident number of ESRD cases from115,829 to 124,114, respectively; (ii) prevalent counts of reported ESRDfrom 592,656 to 703,243, respectively; (iii) incident count ofhemodialysis from 112,804 to 120,972, respectively; and point prevalentcounts for hemodialysis from 414,503 to 495,433 (USRDS 2017). Assuming a<1% incidence and approximately 4% prevalence of calciphylaxis amongpatients on hemodialysis, the estimated annual incident and prevalentcounts for calciphylaxis are around 1200 and 19,800, respectively.Further, according to Mayo Clinic statistics, these numbers representabout 60% of the total U.S. calciphylaxis patient population. The other40% is attributable to CKD patients not on dialysis, which would set thetotal population as of January 2018 at approximately 40,000.

Calciphylaxis is associated with significant morbidity and mortality(Mazhar et al. (2001) “Risk factors and mortality associated withcalciphylaxis in end-stage renal disease, KIDNEY INT. 60(1):324-332;Wilmer et al. (2002) “Emerging Concepts in Prevention, Diagnosis, andTreatment,” SEMIN DIAL. 15(3):172-86; Bhambri et al. (2008)“Calciphylaxis: a review” JOURNAL OF CLINICAL AND AESTHETIC DERMATOLOGY1(2):38-41; Brandenburg et al. (2014), supra; Brandenburg et al. 2016,supra), resulting in a median survival of 1.5 years (Weenig et al.(2007) “Calciphylaxis: natural history, risk factor analysis, andoutcome,” J AM ACAD DERMATOL 56:569-579; Brandenburg et al. (2014),supra). The development of calciphylaxis may be viewed as a two-stageprocess, starting with the development of vascular lesions, followed bytissue ischemia secondary to the vascular lesions. Given that by thetime the clinical entity becomes apparent, it is often too late toreverse the vasculopathy, the prognosis of calciphylaxis is generallypoor (Wilmer et al. (2002), supra). The painful skin ulcers and tissuenecrosis (the hallmarks of calciphylaxis) can lead to serious, and evenlife-threatening or fatal infections, contributing to an overallmortality of up to 80% (Bhambri et al. (2008), supra; Magro et al.(2011) “Calciphylaxis: a review,” JAM COL CERTIF WOUND SPEC. 2(4):66-72;Brandenburg et al. (2016), supra), with more than 50% of patients dyingwithin one year of diagnosis (Mazhar et al. (2001), supra; Bhambri etal. (2008), supra). Apart from death due to sepsis from infected,necrotic skin lesions, fatal internal organ failure has also beenreported in patients with calciphylaxis (Wilmer et al., (2002), supra).In patients with ESRD on hemodialysis, calciphylaxis was found toindependently increase the risk of death by eightfold, and the overall1- and 5-year survival rates have been estimated at 45% and 35%,respectively (Mazhar et al. (2001), supra).

Several in vitro, in vivo and human cohort studies have suggested thatthe use of vitamin K antagonists (VKAs) such as warfarin acceleratescardiovascular calcification (Schurgers et al. (2007a)“Post-translational modifications regulate matrix Gla protein function:importance for inhibition of vascular smooth muscle cell calcification,”J THROMB HAEMOST 5:2503-2511; Schurgers et al. (2007b) “Regression ofwarfarin-induced medial elastocalcinosis by high intake of vitamin K inrats,” BLOOD 109:2823-283; Kruger et al. (2013) “Warfarin inducescardiovascular damage in mice,” ARTERIOSCLER THROMB VASC BIOL 33:2618-2624) and is a prominent risk for calciphylaxis (Hayashi et al.(2012) “A case-control study of calciphylaxis in Japanese end-stagerenal disease patients,” NEPHROL DIAL TRANSPLANT 27:1580-1584; Nigwekaret al. (2013) “Statin use and calcific uremic arteriolopathy: a matchedcase-control study,” AM J NEPHROL 37:325-332; Brandenburg et al. (2016),supra).

In a study evaluating the risk factors for calciphylaxis in hemodialysispatients, 1030 hemodialysis patients with newly diagnosed calciphylaxiswere matched with 2060 controls. Median duration between hemodialysisinitiation and subsequent calciphylaxis development was 925 days(interquartile range [IQR], 273-2185 days). In multivariable conditionallogistic regression analyses, diabetes mellitus; higher body mass index;higher levels of serum calcium, phosphorous, and parathyroid hormone;and nutritional vitamin D, cinacalcet, and warfarin treatments wereassociated with increased odds of subsequent calciphylaxis development(Nigwekar et al. (2016) “A Nationally Representative Study of CalcificUremic Arteriolopathy Risk Factors,” J. AM. SOC. NEPHROL.27(11):3421-9).

Skin necrosis observed in patients with calciphylaxis appears to be dueto extensive small vessel calcification and calcium accumulation in softtissue. The mechanisms for soft tissue calcification are not completelyunderstood; however, carboxylated MGP is considered to play an importantrole as a potent inhibitor of vascular calcification (Schurgers et al.(2007a), supra; Brandenburg et al. (2016), supra; Nigwekar et al.(2016), supra).

Vitamin K is an essential enzymatic co-factor that is required forpost-translational modifications of Vitamin K-dependent (VKD) proteins.A number of VKD proteins are clinically relevant to CKD and ESRDpatients, and include, for example, central coagulation factors such asfactors II, VII, IX, and X and intercellular matrix proteins such asMatrix Gla Protein (MGP) activated protein C and osteocalcin. Vitamin Kis a group of fat soluble vitamins, which include, among other things,vitamin K₁ (also known as phylloquinone), which is made by plants, andvitamin K₂ (also known as menaquinone), which is made by bacteria in gutflora. It is understood that the isoprenoid chain in vitamin K₂ cancontain from 4 to 12 repeating isoprenoid units. For example,menaquinone-4 (or MK-4) contains four isoprenoid units whereasmenaquinone-7 (or MK-7) contains seven isoprenoid units.

With regard to menaquinone-7 (MK-7), under normal conditions MK-7 isreduced to menaquinol-7 (MKH2-7) (a form of Vitamin K hydroquinone) byan NADPH-dependent reductase enzyme or enzymes (e.g., quinoneoxidoreductase). Only the reduced form of MK-7 (namely MKH2-7) functionsas a co-factor for the enzyme gamma glutamate carboxylase (GGCX), whichcatalyzes the carboxylation of Vitamin K-dependent proteins. (See, FIGS.1 and 2.) The enzymatic carboxylation of glutamate residues results inoxidation of MKH2-7 to a 2,3-epoxide form (MK-7 2,3-epoxide). The finalstep of the Vitamin K cycle requires the enzymatic reduction of VitaminMK-7 2,3-epoxide back to MK-7 by Vitamin K epoxide reductase complexsubunit 1 (VKORC1, also referred to as VKOR. In some tissues, theparalog VKORC1L1 (VKORC1-Like-1) may also perform this catalyticreaction. It is known that warfarin blocks both the generation ofMKH2-7, the active form of Vitamin K₂, as well as the regeneration ofMK-7 from Vitamin MK-7 2,3-epoxide, which may lead to the higherincidence of calcification seen among patients receiving warfarintherapy.

Despite efforts to date, there is a need for new clinical approaches toprevent and/or treat calciphylaxis. In particular, there is a need fornew clinical approaches to prevent and/or treat calciphylaxis insubjects with diabetes, CKD, ESRD, and subjects receiving anticoagulantand/or statin therapy.

SUMMARY OF THE INVENTION

It has been discovered that menaquinone-7 (MK-7) and/or menaquinol-7(MKH2-7), can be used effectively at high doses, e.g., in doses of atleast 2 mg per day, to prevent or treat (e.g., slow the progression of,arrest, and/or reverse) calciphylaxis in a subject, e.g., a subject withdiabetes and/or chronic kidney disease.

In one aspect, the disclosure relates to a method of preventing ortreating (e.g., slowing the progression of, arresting, and/or reversing)calciphylaxis in a subject in need thereof. The method includesadministering to the subject an effective amount of a compositioncomprising substantially pure menaquinone-7 (MK-7) and/or menaquinol-7(MKH2-7) thereby to prevent or treat the calciphylaxis. In certainembodiments, the subject has distal calciphylaxis and/or centralcalciphylaxis.

In another aspect, invention provides a method of preventing or treating(e.g., slowing the progression of, arresting, and/or reversing) one ormore symptoms of chronic obstructive pulmonary disease (COPD) in asubject in need thereof, the method comprising administering to thesubject at least 2 mg of substantially pure menaquinone-7 (MK-7) and/ormenaquinol-7 (MKH2-7) per day, thereby to prevent or treat (e.g., slowthe progression of, arrest, and/or reverse) the one or more symptoms ofCOPD, wherein the MK-7 and/or MKH2-7 is administered in a pharmaceuticalcomposition. In certain embodiments, the one or more symptoms isselected from the group consisting of breathing difficulty, cough, mucusproduction, wheezing, and elastinolysis.

In another aspect, the invention provides a disclosure of a method forpreventing or treating (e.g., slowing the progression of, arresting,and/or reversing) tissue calcification in a subject with calciphylaxis,wherein the subject is taking calcium-based phosphate binders and/orvitamin D analogs. The method can include administering to the subjectat least 2 mg of substantially pure menaquinone-7 (MK-7) and/ormenaquinol-7 (MKH2-7) per day. In certain embodiments, the subject hashyperparathyroidism.

In certain embodiments, the pharmaceutical composition comprises MK-7.In certain embodiments, the pharmaceutical composition comprises MKH2-7.In certain embodiments, the pharmaceutical composition comprises acombination of MK-7 and MKH2-7.

In certain embodiments of any of the above aspects, the subject has beendiagnosed as pre-diabetic or has diabetes, e.g., type II diabetes. Incertain embodiments, the subject has chronic kidney disease, e.g., stage3, stage 4, or stage 5. In certain embodiments, the subject has COPD. Incertain embodiments, the subject is undergoing hemodialysis. In certainembodiments, the subject is receiving non-warfarin-based anticoagulanttherapy, such as an oral anti-coagulation therapy. In certainembodiments, if non-warfarin-based anti-coagulation therapy is used, theanti-coagulation therapy can comprise an inhibitor of Factor Xa activity(e.g., apixaban, rivaroxaban, betrixaban, edoxaban, or fondaparinux) orFactor IIa activity (e.g., dabigratran or argatroban). In certainembodiments, the subject has previously been exposed to warfarin-basedanti-coagulation therapy.

In certain embodiments, the subject has a calciphylaxis-related dermallesion. In certain embodiments, administration of the compositionreduces the size of the dermal lesion, for example, administration ofthe composition reduces the total surface area of the dermal lesion byat least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 100%.

In certain embodiments of any of the above aspects, administration ofthe MK-7 and/or MKH2-7 to the subject increases the subject's serum T50value (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) relative to thesubject's serum T50 value prior to administration of the respective MK-7and/or MKH2-7.

In certain embodiments of any of the above aspects, administration ofthe MK-7 and/or MKH2-7 increases a ratio of a carboxylated to anon-carboxylated Vitamin K-dependent protein in the subject's plasma(e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) relative to the ratioprior to administration of the respective MK-7 and/or MKH2-7. In certainembodiments of any of the above aspects, administration of the MK-7and/or MKH2-7 decreases the amount of a non-carboxylated VitaminK-dependent protein in the subject's plasma (e.g., by at least 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100%) relative to the amount prior to administrationof the respective MK-7 and/or MKH2-7. In certain embodiments, theVitamin K-dependent protein is selected from Matrix Gla Protein (MGP),Growth Arrest Specific Gene 6 (Gas-6) protein, PIVKA-II protein,osteocalcin, activated Protein C, activated Protein S, factor II, factorVII, factor IX, and factor X.

In certain embodiments of any of the above aspects, administration ofthe MK-7 and/or MKH2-7 increases the plasma level of osteoprotegerin orFetuin A (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) relative tothe plasma concentration of osteoprotegerin or Fetuin A prior toadministration of the respective MK-7 and/or MKH2-7.

In certain embodiments of any of the above aspects, administration ofthe MK-7 and/or MKH2-7 decreases the plasma level of D-Dimer or HighlySensitive C Reactive Protein (hs-CRP) (e.g., by at least 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100%) relative to the plasma concentration of D-Dimer orHighly Sensitive C Reactive Protein (hs-CRP) prior to administration ofthe respective MK-7 and/or MKH2-7.

In certain embodiments, prior to administration of the composition, thesubject has a uremic oxidative blockade. In certain embodiments, thepresence of a uremic oxidative blockade is determined by measuringincreased plasma lipid peroxidation, e.g., by detection of increased F2isoprostanes (Morrow et al. (1990) “A series of prostaglandin F2-likecompounds are produced in vivo by humans by a non-cyclooxygenase, freeradical-catalyzed mechanism,” PROC. NATL. ACAD. SCI. USA 87:9383-9387),increased isolevuglandin-plasma protein adducts (Salomon et al. (2000)“Isolevuglandin-protein adducts in humans: Products of freeradical-induced lipid oxidation through the isoprostane pathway,”BIOCHIM BIOPHYS ACTA 1485:225-235), increased breath ethane (Handelmanet al. (2000) J AM. SOC. NEPHROL. 11:271A); increased protein and aminoacid oxidation, e.g., by detection of tyrosine residue oxidation(Heinecke et al. (1999) “Detecting oxidative modification ofbiomolecules with isotope dilution mass spectrometry: Sensitive andquantitative assays for oxidized amino acids in proteins and tissues,”METHODS ENZYMOL. 300:124-144), cysteine or methionine residue oxidation,lysine oxidation and threonine oxidation, thiol oxidation and carbonylformation in plasma proteins (Himmelfarb et al. (2000) “Plasma proteinthiol oxidation and carbonyl formation in chronic renal failure,” KIDNEYINT. 58:2571-2578); reactive aldehyde formation, e.g., by detectingglyoxal, methylglyoxal, acrolein, glycoaldehyde, and parahydroxyphenacetaldehyde (Miyata et al. (1999) “Alterations in nonenzymaticbiochemistry in uremia: Origin and significance of ‘carbonyl stress’ inlong-term uremic complications. KIDNEY INT. 55:389-399); increasedreactive carbonyl compounds, e.g., by measuring hydrazine formationafter reaction with 2,4-dinitrophenylhydrazine; diminished plasmaglutathione levels and glutathione peroxidase function (Ceballos-Picotet al. (1996) “Glutathione antioxidant system as a marker of oxidativestress in chronic renal failure,” FREE RADIC. BIOL. MED. 21:845-853);and increased ratio of oxidized to reduced thiols (Hultberg et al.(1995) “Reduced, free, and, total fractions of homocysteine and otherthiol compounds in plasma from patients with renal failure,” NEPHRON70:62-67; Himmelfarb et al. (2002) “Plasma aminothiol oxidation inchronic renal failure,” KIDNEY INT 61:705-716; Ward et al.“Polymorphonuclear leukocyte oxidative burst is enhanced in patientswith chronic renal insufficiency,” JAM. SOC. NEPHROL. 5:1697-1702).

In certain embodiments, the subject is receiving a statin. For example,the statin can be selected from simvastatin, lovastatin, atorvastatin,pravastatin, pitavastatin, rosuvastatin, and fluvastatin.

In certain embodiments of any of the above aspects, the method caninclude administering from about 2 mg to about 1,000 mg of MK-7 and/orMKH2-7 to the subject per day. In other embodiments, the method caninclude administering from about 5 mg to about 1,000 mg of MK-7 and/orMKH2-7 to the subject per day.

In certain embodiments of any of the above aspects, the method caninclude administering from about 2 mg to about 750 mg of MK-7 and/orMKH2-7 to the subject per day. In other embodiments, the method caninclude administering from about 5 mg to about 750 mg of MK-7 and/orMKH2-7 to the subject per day. In certain embodiments of any of theabove aspects, the method can include administering from about 2 mg toabout 500 mg of MK-7 and/or MKH2-7 to the subject per day. In otherembodiments, the method can include administering from about 5 mg toabout 500 mg of MK-7 and/or MKH2-7 to the subject per day. In certainembodiments of any of the above aspects, the method can includeadministering from about 2 mg to about 250 mg of MK-7 and/or MKH2-7 tothe subject per day. In other embodiments, the method can includeadministering from about 5 mg to about 250 mg of MK-7 and/or MKH2-7 tothe subject per day. In certain embodiments of any of the above aspects,the method can include administering from about 2 mg to about 100 mg ofMK-7 and/or MKH2-7 to the subject per day. In other embodiments, themethod can include administering from about 5 mg to about 100 mg of MK-7and/or MKH2-7 to the subject per day. In other embodiments, the methodcan include administering from about 10 mg to about 75 mg of MK-7 and/orMKH2-7 to the subject per day, e.g., administering 10, 25, 50 or 75 mgof MK-7 and/or MKH2-7 to the subject per day.

In certain embodiments, the MK-7 and/or MKH2-7 is administered to thesubject for at least 2 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 6months, 1 year, or indefinitely. If the subject is undergoinghemodialysis, the MK-7 and/or MKH2-7 can be administered to the subjectfor a period that includes at least the duration of hemodialysis.

In certain embodiments, the MK-7 and/or MKH2-7 is administered orally.The MK-7 and/or MKH2-7 can be disposed within a tablet, caplet orcapsule. The MK-7 and/or MKH2-7 can be present in a compositioncomprising a pharmaceutically acceptable excipient.

The description above describes multiple aspects and embodiments of theinvention. The patent application specifically contemplates allcombinations and permutations of the aspects and embodiments. These andother aspects and features of the invention are described in thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of theinvention will become apparent from the following description ofpreferred embodiments, as illustrated in the accompanying drawings. Likereferenced elements identify common features in the correspondingdrawings, in which:

FIG. 1 is a schematic diagram depicting the Vitamin K cycle and theeffects of uremia and hemodialysis-induced oxidation of Vitamin Khydroquinone; and

FIG. 2 is a schematic diagram depicting the carboxylation of VitaminK-dependent proteins by gamma-glutamate carboxylase (GGCX).

DETAILED DESCRIPTION

The invention is based, in part, upon the discovery that menaquinone-7(MK-7) and/or menaquinol-7 (MKH2-7), the reduced form of MK-7, can beadministered to a subject in need thereof to prevent or treat (e.g.,slow the progression of, arrest, and/or reverse) calciphylaxis in thesubject, e.g., a subject with diabetes and/or chronic kidney disease.

Without wishing to be bound by the theory, it is believed that oxidativestress induced by the retention of uremic toxins depletes vascularendothelium of functional Vitamin K₂ leading to suboptimalconcentrations of certain Vitamin K-dependent proteins, includingcarboxylated MGP and activated Protein C. The subsequent reduced calciumbinding capacity of these and other vitamin-dependent proteinscontributes to abnormal tissue calcification. High doses ofsubstantially pure MK-7 and/or MKH2-7 can be used to increasecarboxylation of Vitamin K-dependent proteins, thereby preventing ortreating (e.g., slowing the progression of, arresting, and/or reversing)calciphylaxis, wherein the MK-7 and/or MKH2-7 are administered in theform of a pharmaceutical composition. The MK-7 and/or MKH2-7 can beadministered in a single dosage unit (for example, a single capsule) orin multiple dosage units (for example, multiple capsules) provided thatthe requisite amount of MK-7 and/or MKH2-7 is administered per day. Incertain embodiments, the pharmaceutical composition comprises MK-7. Incertain embodiments, the pharmaceutical composition comprises MKH2-7. Incertain embodiments, the pharmaceutical composition comprises acombination of MK-7 and MKH2-7.

I. Menaquinone-7 (MK-7) and/or Menaquinol (MKH2-7)

The compositions of the invention comprise menaquinone-7 (MK-7), a formof Vitamin K₂. The IUPAC name for MK-7 is as2-[(2E,6E,10E,14E,18E,22E)-3,7,11,15,19,23,27-heptamethyloctacosa-2,6,10,14,18,22,26-heptaenyl]-3-methylnaphthalene-1,4-dione,and the chemical structure of MK-7 is shown in Formula I.

Also contemplated for use in accordance with the methods herein ismenaquinol-7 (MKH2-7), which is a reduced form of MK-7. The IUPAC namefor MKH2-7 is2-[(2E,6E,10E,14E,18E,22E)-3,7,11,15,19,23,27-heptamethyloctacosa-2,6,10,14,18,22,26-heptaenyl]-3-methylnaphthalene-1,4-diol,and the chemical structure of MKH2-7 is set forth in Formula

Without wishing to be bound by the theory, it is believed thatsupplemental MK-7 and/or MKH2-7 forms of Vitamin K provide increasedprotection against non-specific oxidation of Vitamin K intermediatescaused by uremia and/or dialysis.

The long aliphatic chain of MK-7 is not synthesized by humans but issynthesized in the colon by bacteria. Also, dietary sources of MK-7include bacterially fermented foods such as nattō (soy beans fermentedby Bacillus subtilis), cheeses, sauerkraut and buttermilk and pork, eel,plaice, and buckwheat bread. In addition, MK-7 is available as anutritional supplement (e.g., Vitamin K2 MK-7 from RejuvenationTherapeutics®; Bio-Tech™ Pharmacal MK-7 Vitamin K2, Fayetteville Ark.;NOW® Foods MK-7 Vitamin K-2).

The invention, however, employs substantially pure MK-7 and/or MKH2-7,administered in the form of a pharmaceutical composition.

MK-7 can also be produced synthetically, for example, as described inBaj et al. (2016) “Convergent Synthesis of Menaquinone-7 (MK-7),” ORG.PROCESS RES. DEV. 20:1026-1033, WO2010/034999 and WO2010/035000 (alsopublished as U.S. Patent Application No. 2011/0207967).

MKH2-7 can be synthesized from MK-7 by reducing MK-7 using conventionalreduction reactions known in the art, including, for example, byreduction with zinc and acetic acid as described by Marchand et al.(1991) “Mild and Highly Selective Ultrasound-promoted Zinc/Acetic AcidReduction of C═C Bonds in α,β-Unsaturated γ-Dicarbonyl Compounds,”SYNTHESIS 1991(3):198-200.

In this method, MK-7 (e.g., 1 g, 1.54 mmol) is dissolved in acetic acidglacial (15 mL) and powdered zinc (e.g., 0.8 g, 12.3 mmol) is added. Theresulting mixture is sonicated for 0.5 hour or the reaction can berefluxed until completion. The resulting mixture is filtered and theresidue is washed with dichloromethane. The combined filtrates areconcentrated in vacuo, to produce the pure reduction product. Theprocedure is performed under Argon. To stabilize MKH2-7 in the reducedform it may be helpful to admix MKH2-7 with one or more antioxidantssuch as vitamin C, a vitamin C ester (e.g., ascorbyl palmitate), and/orvitamin E, e.g., within a capsule or softgel. Alternatively, prodrugs ofMKH2-7 can be created, for example, where one or both of the hydroxylgroups are esterified with various groups (for example, acetate) toproduce a stable prodrug that is metabolized to produce MKH2-7 in thesubject.

II. Dosage Forms and Administration

In certain embodiments, compositions useful in accordance with theinvention comprise, consist essentially of, or consist of substantiallypure MK-7 and/or MKH2-7. Substantially pure refers to a composition ofactive ingredient comprising at least 95%, at least 96%, at least 97%,at least 98%, at least 99% or at least 99.5% by weight MK-7 and/orMKH2-7. In certain embodiments, MK-7 and/or MKH2-7 are the sole vitaminsadministered in a dosage form.

As used herein, the phrases “effective amount” and “therapeuticallyeffective amount” refer to the amount of a compound (e.g., MK-7 and/orMKH2-7) sufficient to effect one or more beneficial or desired results.An effective amount can be administered in one or more administrations,applications or dosages and is not intended to be limited to aparticular formulation or administration route.

In another embodiment, the invention provides a dosage form, forexample, an oral dosage form, comprising an effective amount of MK-7and/or MKH2-7, for example, from about 2 mg to about 1,000 mg, fromabout 2 mg to about 750 mg, from about 2 mg to about 500 mg, from about2 mg to about 250 mg, from about 2 mg to about 200 mg, from about 2 mgto about 150 mg, from about 2 mg to about 100 mg, from about 2 mg toabout 50 mg, from about 2 mg to about 25 mg, from about 5 mg to about1,000 mg, from about 5 mg to about 750 mg, from about 5 mg to about 500mg, from about 5 mg to about 250 mg, from about 5 mg to about 200 mg,from about 5 mg to about 150 mg, from about 5 mg to about 100 mg, fromabout 5 mg to about 50 mg, from about 5 mg to about 25 mg, from about 10mg to about 1,000 mg, from about 10 mg to about 750 mg, from about 10 mgto about 500 mg, from about 10 mg to about 250 mg, from about 10 mg toabout 200 mg, from about 10 mg to about 150 mg, from about 10 mg toabout 100 mg, from about 10 mg to about 50 mg, from about 10 mg to about25 mg, from about 15 mg to about 1,000 mg, from about 15 mg to about 750mg, from about 15 mg to about 500 mg, from about 15 mg to about 250 mg,from about 15 mg to about 200 mg, from about 15 mg to about 150 mg, fromabout 15 mg to about 100 mg, from about 15 mg to about 50 mg, from about15 mg to about 25 mg, from about 25 mg to about 1,000 mg, from about 25mg to about 750 mg, from about 25 mg to about 500 mg, from about 25 mgto about 250 mg, from about 25 mg to about 200 mg, from about 25 mg toabout 150 mg, from about 25 mg to about 100 mg, from about 25 mg toabout 50 mg, from about 50 mg to about 1,000 mg, from about 50 mg toabout 750 mg, from about 10 mg to about 500 mg, from about 50 mg toabout 250 mg, from about 50 mg to about 200 mg, from about 50 mg toabout 150 mg, from about 50 mg to about 100 mg, from about 75 mg toabout 1,000 mg, from about 75 mg to about 750 mg, from about 75 mg toabout 500 mg, from about 75 mg to about 250 mg, from about 75 mg toabout 200 mg, from about 75 mg to about 150 mg, from about 75 mg toabout 100 mg, from about 100 mg to about 1,000 mg, from about 100 mg toabout 750 mg, from about 100 mg to about 500 mg, from about 100 mg toabout 250 mg, from about 100 mg to about 200 mg, or from about 100 mg toabout 150 mg. In certain embodiments, 2 mg, 5 mg, 10 mg, 25 mg, 75 mg or100 mg of substantially pure MK-7 and/or MKH2-7 is administered to thesubject in a suitable dosage form, such as a tablet, caplet or capsule(e.g., a liquid or gel capsule).

In certain embodiments, the methods can include administering from about2 mg to about 100 mg of MK-7 and/or MKH2-7 to the subject per day. Inother embodiments, the method can include administering from about 2.5mg to about 100 mg, from about 3 mg to about 100 mg, from about 4 mg toabout 100 mg, or from about 5 mg to about 100 mg of MK-7 and/or MKH2-7to the subject per day. In certain embodiments, the method can includeadministering about 2 mg, 2.5 mg, about 3 mg, about 4 mg, about 5 mg,about or 7.5 mg of MK-7 and/or MKH2-7 to the subject per day. In otherembodiments, the method can include administering from about 10 mg toabout 100 mg of MK-7 and/or MKH2-7 to the subject per day, e.g.,administering 10, 25, 50, 75 or 100 mg of MK-7 and/or MKH2-7 to thesubject per day.

It is understood that the MK-7 and/or MKH2-7 can be administered in asingle dosage unit (e.g., 1 capsule) or in multiple (e.g., 2, 3, or 4,etc.) dosage units (e.g., multiple capsules). A composition for use inaccordance with the invention can be formulated as one or more dosageunits. Such dosage units may be administered once a day or a plurality(e.g., 1 to about 10, 1 to about 8, 1 to about 6, 1 to about 4 or 1 to2) of times per day, or as many times as needed to elicit a therapeuticresponse. The dosage units can be packaged in a kit, for example, a kitcontaining one or more blister packages of about 1 to about 20 dosageunits (e.g., capsules) per sheet or a bottle containing a plurality ofdosage units (e.g., capsules).

In certain embodiments, the pharmaceutical composition containing theMK-7 and/or MKH2-7 can be formulated for administration in solid orliquid form, including drenches (aqueous or non-aqueous solutions orsuspensions), tablets (e.g., those targeted for buccal, sublingual,and/or systemic absorption), boluses, powders, granules, pastes forapplication to the tongue, and/or topical creams. As used herein, theterm “pharmaceutical composition” refers to the combination of an activeagent with one or more pharmaceutically acceptable carriers (inert oractive) making the composition especially suitable for diagnostic ortherapeutic use in vivo or ex vivo. As used herein, the term“pharmaceutically acceptable carrier” refers to any of the standardpharmaceutical carriers and excipients, such as a phosphate bufferedsaline solution, water, emulsions (e.g., such as an oil/water orwater/oil emulsions), and various types of wetting agents. Thecompositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers and adjuvants, see Martin, Remington'sPharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, Pa. (1975).

In certain embodiments, the MK-7 and/or MKH2-7 composition can beformulated in an oil, such as castor oil, sesame oil, medium chaintriglyceride (MCT) oil, olive oil, soybean oil, or coffee bean oil.Excipients suitable for use with the MK-7 and/or MKH2-7 compositioninclude antioxidants, bioavailability enhancers, solubility enhancers orsolubilizers, stabilizers, etc.

In certain embodiments, the MK-7 and/or MKH2-7 composition includes oneor more antioxidants such as one or more antioxidants selected fromDL-alpha tocopherol (vitamin E), ascorbic acid (vitamin C) or a vitaminC ester, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA),lecithin, citric acid, sesame oil, olive oil, soybean oil, and coffeebean oil.

In certain embodiments, the MK-7 and/or MKH2-7 composition includes anemulsifier selected from Tween® 20 (polyethylene glycol sorbitanmonolaurate), Tween® 80 (polyethylene glycol sorbitan monooleate),Kolliphor® EL (polyoxyl castor oil, also called Cremophor EL), Capmul®MCM (mono-diglyceride of medium chain fatty acids such as caprylic andcapric), lecithin, Lauroglycol™ 90 (propylene glycol monolaurate (typeII)), deoxycholic acid, Phosal® 50 PG (lecithin in propylene glycol,sunflower mono-diglycerides, ascorbyl palmitate), beeswax, and waxflakes.

In certain embodiments, the MK-7 and/or MKH2-7 composition includes abioavailability enhancer selected from Maisine® CC (glycerylmonolinoleate), Gelucire® 44/14 (lauroyl polyoxyl-32 glycerides),Capmul® PG-8 NF (propylene glycol monoester of caprylic acid), Plurol®Oleique (polyglyceryl-6 dioleate), Lauroglycol™ 90 (propylene glycolmonolaurate (type II), Labrasol® (caprylocaproyl polyoxyl-8 glycerides),Captisol® (SBE-beta-cyclodextrin), Labrafil® M2125CS (linoleoylPolyoxyl-6 glycerides), and Phosal® 50 PG (lecithin in propylene glycol,sunflower mono-diglycerides, ascorbyl palmitate).

In certain embodiments, the MK-7 and/or MKH2-7 composition includes asolubility enhancer or solubilizer selected from oleic acid, Kolliphor®EL (polyoxyl castor oil, also called Cremophor EL), Vitamin E TPGS(D-α-tocopherol polyethylene glycol-1000 succinate), Maisine® CC(glyceryl monolinoleate), Gelucire® 44/14 (lauroyl polyoxyl-32glycerides), Miglyol® 812N (esters of saturated coconut and palm kerneloil-derived caprylic fatty acids and glycerin), Plurol® Oleique(Polyglyceryl-6 Dioleate), Lauroglycol™ 90 (propylene glycol monolaurate(type II), Labrasol® (Caprylocaproyl polyoxyl-8 glycerides), Kolliphor®EL (polyoxyl castor oil), Captisol® (SBE-beta-cyclodextrin), Encapsin™HPB (hydroxypropyl-beta-cyclodextrin), Peceol™ (glycerol/glycerylmonooleate (type 40)), sodium deoxycholate, deoxycholic acid, Labrafil®M2125CS (linoleoyl Polyoxyl-6 glycerides), and medium-chain mono- anddiglycerides.

In certain embodiments, the MK-7 and/or MKH2-7 composition includes asolvent selected from the group consisting of isopropyl myristate,Capmul® PG-8 NF (propylene glycol monoester of caprylic acid),Lauroglycol™ 90 (propylene glycol monolaurate (type II)), Polyethyleneglycol (PEG), and propylene glycol.

In certain embodiments, the MK-7 and/or MKH2-7 composition includes astabilizer selected from the group consisting of oleic acid and DL-alphatocopherol.

In certain embodiments, the MK-7 and/or MKH2-7 composition includes asurfactant selected from the group consisting of sodium oleate, Span 20(sorbitan laurate), Span® 80 (sorbitan oleate), Vitamin E TPGS(D-α-tocopherol polyethylene glycol-1000 succinate), Lauroglycol™ 90(propylene glycol monlaurate (type II)), Labrasol® (Caprylocaproylpolyoxyl-8 glycerides), polyethylene glycol (PEG), and Captisol®(SBE-beta-cyclodextrin).

Other suitable excipients include one or more fatty acids selected fromlauric, valeric, caproic, capric, caprylic, myristic, palmitic,palmitoleic, stearic, and arachidic acid and their esters.

In certain embodiments, compositions useful in the practice of theinvention are orally deliverable. The terms “orally deliverable” or“oral administration” herein include any form of delivery of thecompositions described herein to a subject, wherein the composition isplaced in the mouth of the subject, whether or not the composition isswallowed. Thus “oral administration” includes buccal and sublingual aswell as esophageal administration.

In certain embodiments, the MK-7 and/or MKH2-7 is administered to thesubject for at least 2 weeks, e.g., 2, 3, 4, 5 weeks, or more. Incertain embodiments, the MK-7 and/or MKH2-7 is administered for at least6 weeks. In certain embodiments, the MK-7 and/or MKH2-7 is administeredto a subject for at least 2 months, at least 3 months, at least 6months, at least 1 year, at least 2 years, at least 3 years, orindefinitely. In certain embodiments, the MK-7 and/or MKH2-7 isadministered at least as long as the subject is receiving ananticoagulant therapy that is not warfarin or a drug having a similarmechanism of action as warfarin (e.g., acenocoumarol and phenprocoumon)and/or statin therapy. If the subject is undergoing hemodialysis, theMK-7 and/or MKH2-7 can be administered to the subject for a period thatincludes the duration of hemodialysis.

In certain embodiments, the MK-7 and/or MKH2-7 composition isadministered together with a statin such as simvastatin, lovastatin,atorvastatin, pravastatin, pitavastatin, rosuvastatin, and fluvastatin.The MK-7 and/or MKH2-7 composition can be formulated into a singledosage form, such as a dosage form discussed herein, with the statin. Inother embodiments, the MK-7 and/or MKH2-7 composition is formulated intoa separate dosage form from the statin. Formulation of statins, such assimvastatin, lovastatin, atorvastatin, pravastatin, pitavastatin,rosuvastatin, and fluvastatin, is well known in the art.

As used herein, the terms “subject” and “patient” are usedinterchangeably and refer to an organism to be treated by the methodsand compositions of the present invention. Such organisms are preferablymammals (e.g., human, mouse, rat, guinea pig, dog, cat, horse, cow, pig,or non-human primate, such as a monkey, chimpanzee, baboon, and rhesus),and more preferably humans.

III. Methods

In certain aspects, the invention provides a method of preventing ortreating (e.g., slowing the progression of, arresting, and/or reversing)calciphylaxis in a subject in need thereof. In certain embodiments, themethod relates to preventing or slowing the progression ofcalciphylaxis. In certain embodiments, the method relates to arrestingthe progression of calciphylaxis. In certain embodiments, the methodrelates to reversing calciphylaxis.

The method can include administering to the subject a menaquinone-7(MK-7) and/or menaquinol-7 (MKH2-7) composition as described herein(e.g., at least 2 mg of substantially pure MK-7 and/or MKH2-7 per day,e.g., 10, 15, 25, 50 or 75 mg per day), thereby to prevent, slow theprogression of, or reverse calciphylaxis. In certain embodiments, thesubject has distal calciphylaxis (i.e., affecting the limbs) and/orcentral calciphylaxis (i.e., affecting the thorax).

In certain aspects, the invention provides a method of improving aorticcompliance in a subject with calciphylaxis, the method comprisingadministering to the subject an effective amount of substantially puremenaquinone-7 (MK-7) and/or menaquinol-7 (MKH2-7) per day. In certainembodiments, the invention provides a method of preventing or treating(e.g., slowing the progression of, arresting, and/or reversing)peripheral vasculopathy in a subject in need thereof, wherein thesubject has ESRD or CKD, the method comprising administering to thesubject an effective amount of substantially pure menaquinone-7 (MK-7)and/or menaquinol-7 (MKH2-7) per day.

As used herein, the term “treating” includes any effect, for example,lessening, reducing, modulating, arresting, slowing the progression of,ameliorating or eliminating, that results in the improvement of thecondition, disease, disorder, and the like, or ameliorating a symptomthereof. Treating can be curing, improving, or at least partiallyameliorating the disorder. In certain embodiments, treating is curingthe disease. The term “disorder” refers to and is used interchangeablywith, the terms disease, condition, or illness, unless otherwiseindicated.

1. Diabetes, CKD, ESRD, Hemodialysis

In certain embodiments, the subject to be treated with the methodsand/or compositions described herein has been diagnosed as pre-diabeticor has diabetes, e.g., type I or II diabetes. Diabetes often leads tochronic kidney disease (CKD) due to uncontrolled blood sugar and bloodpressure damaging capillaries in the kidney. CKD often leads to thedevelopment of uremia (urea in the blood), which can inhibit cyclicregeneration of Vitamin K, leading to calciphylaxis. Accordingly, incertain embodiments, the subject has CKD.

Patients with chronic kidney disease are classified into stages basedupon their glomerular filtration rate (GFR). The GFR for stage 1patients is >90 mL/minute/1.73 m², stage 2 is 60-89 mL/minute/1.73 m²,stage 3 is 30-59 mL/minute/1.73 m², stage 4 is 15-29 mL/minute/1.73 m²,and stage 5 is <15 mL/minute/1.73 m². In certain embodiments, thesubject has stage 3, stage 4, or stage 5 CKD. Patients with stage 4 or 5CKD are considered to have end stage renal disease (ESRD). Other typesof kidney disease, such as polycystic kidney disease, also can lead toESRD. In certain embodiments, the subject has ESRD.

Patients with CKD and/or ESRD often require hemodialysis. However,hemodialysis can lead to the oxidation of numerous tissue proteins, andboth CKD and ESRD patients often exhibit a higher percentage (15-fold)of carbonyl proteins compared to normal controls. The percentage ofcarbonyl proteins may be even higher among patients receivinghemodialysis, suggesting that hemodialysis contributes to oxidativeburden.

As discussed supra, the oxidative load generated by hemodialysis isbelieved to lead to the oxidation of MKH2-7 back to MK-7. It iscontemplated that the oxidation of Vitamin K hydroquinone (KH2) byhemodialysis blocks its ability to function as a co-factor for GGCXleading to reduced gamma carboxylation of Vitamin K-dependent proteins.Thus, the oxidative effects of hemodialysis may contribute to thecalciphylaxis observed within the CKD and ESRD populations. Accordingly,in certain embodiments herein, the subject is receiving hemodialysis.

2. Anticoagulant Therapy

Vitamin K is necessary for the production of clotting factors. However,certain anticoagulants, for example, certain oral anticoagulants, act asVitamin K antagonists to prevent blood clotting. Given thatcalciphylaxis can occur when Vitamin K activity is dysregulated, themethods herein relate to preventing or treating (e.g., slowing theprogression of, arresting, and/or reversing) calciphylaxis in a subjectreceiving anticoagulant therapy, and in need thereof, comprisingadministering to the subject a menaquinone-7 (MK-7) and/or menaquinol-7(MKH2-7) containing composition as described herein, thereby to preventor treat calciphylaxis.

Exemplary anti-coagulant therapies include inhibitors of Factor Xaactivity or Factor Ha activity. Inhibitors of Factor Xa activity includethe oral therapies apixaban (e.g., Eliquis®, Bristol-Myers Squibb),rivaroxaban (e.g., Xarelto®, Janssen), betrixaban (e.g., Bevyxxa®,Portola Pharmaceuticals), and edoxaban (e.g., Savaysa®, Daiichi Sankyo),and the subcutaneous therapy fondaparinux (e.g., Arixtra®,GlaxoSmithKline). Inhibitors of Factor IIa (thrombin) activity includethe oral therapies dabigratran (e.g., Pradaxa®, Boehringer Ingelheim)and the intravenous therapy argatroban (e.g., Pfizer).

In certain embodiments, the subject is simultaneously receiving anon-warfarin-based anticoagulant therapy. Non-warfarin-basedanticoagulant therapies can be based on an anticoagulant that does notprimarily act to block the conversion of Vitamin K to Vitamin Khydroquinone and/or the conversion of Vitamin K 2,3-epoxide back toVitamin K. Exemplary non-warfarin-based anticoagulants may act as ainhibitor of Factor Xa activity or an inhibitor of Factor IIa activity.Exemplary non-warfarin-based anticoagulants include apixaban,rivaroxaban, betrixaban, edoxaban, dabigratran, fondaparinux, andargatroban. In certain embodiments, the subject has previously beenexposed to warfarin-based anti-coagulation therapy.

In certain embodiments, the methods described herein relate topreventing, slowing the progression of, arresting, and/or reversingcalciphylaxis in a subject in need thereof, wherein the subject hasstage 5 CKD and is undergoing oral, non-warfarin-based anticoagulanttherapy. The method can comprise administering to the subject at least 2mg of substantially pure menaquinone-7 (MK-7) and/or menaquinol-7(MKH2-7) per day, thereby to prevent or treat (e.g., slow theprogression of, arrest, and/or reverse) calciphylaxis. The subject mayalso have diabetes and/or may be undergoing hemodialysis.

3. Statin Therapy

It has been observed that coronary artery calcification can be increasedupon statin use (Saremi et al. (2012) “Progression of VascularCalcification Is Increased With Statin Use in the Veterans AffairsDiabetes Trial (VADT),” DIABETES CARE 35:2390-2392). Lipophilic statinshave been shown to inhibit the enzymatic activity of UbiAprenyltransfease domain-containing protein (UBIAD1), an enzyme thatplays a role in Vitamin K synthesis (Nakagawa et al. (2010)“Identification of UBIAD1 as a novel human menaquinone-4 biosyntheticenzyme,” NATURE 468(7320):117-21). Further, in vitro experiments appearto demonstrate that Vitamin K synthesis is impaired in the presence ofstatins (Chen et al. (2017) “Does statins promote vascular calcificationin chronic kidney disease?” EUR. J. CLIN. INVEST. 47(2): 137-148).

Without wishing to be bound by the theory, it is contemplated that HMGcoA reductase inhibitors directly inhibit MK-4 production in coronaryvascular smooth muscle, which may explain why statins have not beenshown to reduce cardiovascular mortality in CKD or ESRD patients. Onepossible explanation for this observation is that CDK and ESRD patientsare functionally Vitamin K deficient, and that blocking endogenousvascular smooth muscle cell vitamin K (MK-4) production only serves toworsen the calcification of the media of the vessel, thus mitigating anypotential benefits of lipid reduction. Moreover, the loss of vascularcompliance accompanying the increase in calcification may contribute tooverall cardiovascular mortality.

Accordingly, provided herein is a method for preventing or treating(e.g., slowing the progression of, arresting, and/or reversing)calciphylaxis in a subject in need thereof, wherein the subject isreceiving a statin. The method can include administering to the subjecta menaquinone-7 (MK-7) and/or menaquinol-7 (MKH2-7) composition asdescribed herein. In certain embodiments, the subject is receiving astatin, such as simvastatin (e.g., Zocor®, Merck & Co., Inc.),lovastatin (e.g., Mevacor®, Merck & Co., Inc.), atorvastatin (e.g.,Lipitor®, Pfizer), pravastatin (e.g., Pravachol®, Bristol-Myers SquibbCo.), pitavastatin (e.g., Livalo®, Kowa Pharmaceuticals America),rosuvastatin (e.g., Crestor®, AstraZeneca), and fluvastatin (e.g.,Lescol®, Novartis Pharmaceuticals).

Further provided herein is a method for preventing or treating (e.g.,slowing the progression of, arresting, and/or reversing) calciphylaxisin a subject in need thereof, wherein the method includes administeringto the subject menaquinone-7 (MK-7) and/or menaquinol-7 (MKH2-7)composition as described herein and a statin, such as simvastatin,lovastatin, atorvastatin, pravastatin, pitavastatin, rosuvastatin, andfluvastatin. The menaquinone-7 (MK-7) and/or menaquinol-7 (MKH2-7) andstatin can be administered as separate dosage forms, or in the samedosage form.

4. Improving Aortic Compliance, and Arresting and/or ReversingPeripheral Vasculopathy

In certain embodiments, the disclosure relates to a method of arrestingand/or reversing peripheral vasculopathy in a subject in need thereof,wherein the subject has ESRD or CKD, the method comprising administeringto the subject an effective amount of substantially pure menaquinone-7(MK-7) and/or menaquinol-7 (MKH2-7). In certain embodiments, thedisclosure relates to a method of improving aortic compliance in asubject in need thereof, the method comprising administering to thesubject an effective amount of substantially pure menaquinone-7 (MK-7)and/or menaquinol-7 (MKH2-7).

The term “vasculopathy” refers to any disease or disorder affectingblood vessels. It can include any inflammatory, metabolic, coagulative,embolic or degenerative disease, disorder, or condition. In certainembodiments, the vasculopathy is vascular calcification, e.g., medial orintimal vascular calcification.

One of the clinical consequences of vascular calcification is arterialstiffness, which leads to a decrease in vascular compliance (e.g.,vascular elasticity), e.g., in aortic compliance. Thus, in accordancewith the methods disclosed herein, administration of an effective amountof substantially pure menaquinone-7 (MK-7) and/or menaquinol-7 (MKH2-7)may improve aortic compliance. Reduction in aortic compliance can beassessed using aortic plethysmography (i.e., the capacity of a bloodvessel to dilate), see, e.g., Inuma et al. (2012) HONG KONG JOURNAL OFNEPHROLOGY 14(2):48-53. Reduction in aortic compliance also can beassessed by measuring pulse wave velocity (PWV). PWV is the velocity atwhich the arterial pulse moves through the circulatory system and isused as a measure of arterial stiffness. Higher PWV corresponds tohigher arterial stiffness, and therefore lower vascular compliance.Methods for measuring PWV are known in the art and described, forexample, by Pereira et al. (2015) “Novel Methods for Pulse Wave VelocityMeasurement,” J. MED. BIOL. ENG. 35:555-565. Briefly, PWV can beassessed by measuring regional PWV, such as carotid-femoral PWV, orlocal PWV. Methods for measuring regional PWV are known in the art andinclude, for example, the use of a pulse transducer probe, TY-360pressure transducer, doppler unit synchronism with electrocardiogram(ECG), pulse transducer probes, photoplethysmography, Complior®(piezoelectric pressure transducers), SphygmoCor®, Arteriograph®, andPulsePen® (tonometer and integrated electrocardiogram unit). Id. Methodsfor measuring local PWV are known in the art and include, for example,magnetic resonance imaging, ultrasound, and angiography. Id.

In certain embodiments, administration of the MK-7 and/or MKH2-7increases aortic compliance of a blood vessel by at least 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100%. In certain embodiments, administration of the MK-7and/or MKH2-7 increases aortic compliance of a blood vessel by about10%-500%, by about 50%-200%, by about 75%-125%. In certain embodiments,administration of the MK-7 and/or MKH2-7 increases aortic compliance ofa blood vessel by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to avessel prior to administration of the MK-7 and/or MKH2-7. In certainembodiments, administration of the MK-7 and/or MKH2-7 increases aorticcompliance of a blood vessel by about 10%-500%, by about 50%-200%, byabout 75%-125% relative to a vessel prior to administration of the MK-7and/or MKH2-7.

Coronary arterial calcium scores (CAC) can be used to estimate theextent of calcification of thoracic arteries. A high CAC score isindicative of calcification, and treatment has the aim of arresting thelong term increase in CAC score, or reversing it, or slowing the rate ofincrease.

5. Chronic Obstructive Pulmonary Disease

Chronic Obstructive Pulmonary Disease is a term used to describeprogressive lung disease that makes breathing difficult. The two primaryforms of COPD are emphysema and chronic bronchitis. Symptoms of COPDinclude difficulty breathing, cough, mucus production, and wheezing. Inaddition, elastinolysis (proteolysis of elastin) is a key feature ofCOPD. It contributes to the loss of arterial flexibility and promotescalcification of the intimal media of blood vessels. It also has beenshown to be a strong predictor of mortality in COPD patients (Rabinovichet al., (2016) “Circulating desmosine levels do not predict emphysemaprogression but are associated with cardiovascular risk and mortality inCOPD,” ERJ Express doi: 10.1183/13993003.01824-2015). MGP has beendemonstrated to inhibit the production of matrix metalloproteases thatpromote elastinolysis. Therefore, it has been hypothesized that VitaminD is a critical determinant of the rate of elastin degradation, and thatlow Vitamin D levels lead to low MGP activity that is inadequate toprotect from elastinolysis (Piscaer et al., (2017) “Vitamin Ddeficiency: the linking pin between COPD and cardiovascular diseases?”RESP. RES. 18:189). Without wishing to be bound by the theory, enhancedproduction of activated (carboxylated) MGP by administration of MK-7and/or MKH2-7 can act to suppress the deleterious effects ofelastinolysis in a subject having COPD thereby to prevent, or slow theprogression of, or reverse the one or more symptoms of COPD.

Reduction of symptoms of COPD, including difficulty breathing, cough,mucus production, and wheezing, can be measured by any means known inthe art. A slowing of the rate of increase of one or more COPD symptomsover time is indicative of a slowing of the progression of the one ormore symptoms of COPD, or a reduction in one or more symptoms, such asby 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 100% is indicative of a reversal of the oneor more symptoms of COPD.

Elastinolysis can be measured by any method known in the art, including,for example, by measuring blood isodesmosine levels in a subject. Aslowing of the rate of increase in isodesmosine levels over time isindicative of a slowing of the progression of the one or more symptomsof COPD, or a reduction in isodesmosine levels, such as by 5%, 10%, 20%,25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90% or 100% is indicative of areversal of the one or more symptoms of COPD.

6. Hyperparathyroidism

The incidence of calciphylaxis appears to be increasing, which may bedue, in part, to the administration of calcium-based phosphate bindersand vitamin D analogs, e.g., for the treatment of hyperparathyroidism.(Goel et al. (2011) “Treatment of Severe Metastatic Calcification andCalciphylaxis in Dialysis Patients,” INTERNATIONAL JOURNAL OF NEPHROLOGY2011:1-5.) Accordingly, in one aspect, the invention provides adisclosure of a method for preventing calciphylaxis in a subject orslowing the progression of, arresting, and/or reversing tissuecalcification in a subject with calciphylaxis, wherein the subject istaking calcium-based phosphate binders and/or vitamin D analogs. Themethod can include administering to the subject at least 2 mg ofsubstantially pure menaquinone-7 (MK-7) and/or menaquinol-7 (MKH2-7) perday. In certain embodiments, the subject has hyperparathyroidism.

Examples of calcium-based phosphate binders include calcium carbonateand calcium acetate. Examples of vitamin D analogs include calcitrol,alfacalcidol, paricalcitol, maxacalcitol, and2-methylene-19-nor-(20S)-1α,25(OH)₂D₃ (2MD).

IV. Biomarkers Useful for Determining Prevention, Slowing theProgression of, Arresting, and/or Reversing, Calciphylaxis in a Subject

In certain embodiments, biomarkers can be assessed to determine whetherthe methods and/or compositions described herein are effective atpreventing or treating (e.g., slowing the progression of, arresting,and/or reversing) calciphylaxis in a subject. These include, forexample, measuring a subject's T50 times and measuring a ratio ofcarboxylated Vitamin K-dependent proteins to non-carboxylated proteins,as described in more detail below.

1. Primary Calciprotein Particles (CPPs) and Serum T50

Under normal physiologic conditions, pathological calcification isprevented in part by primary calciprotein particles (CPPs) (Price, etal., supra). CPPs comprise circulating calcium phosphate crystalscomplexed with two calcification inhibiting proteins, Fetuin A andMatrix Gla Protein. (Id.) Matrix Gla Protein is a Vitamin K-dependentprotein and formation of CPPs is dependent upon the gamma carboxylationof Matrix Gla Protein. (Id.) Pre-clinical studies suggest that thecalciprotein system functions as an alternative means for preventingpathologic calcification when humoral lines of defense such aspyrophosphate, magnesium and albumin are overwhelmed. The binding ofcalcium-phosphate crystals by primary CPPs occurs in a coordinated andtime-dependent process.

The time necessary for 50% (T50) conversion of primary CPPs to secondaryCPPs is an accurate and highly sensitive means for determining thecapacity of plasma to sequester excess calcium phosphate crystals. Theconversion from primary to secondary forms involves the formation of amore elongated crystal. The elongation leads to a more turbid solutionwhich can be detected by nephelometry. Under conditions of heavy calciumphosphate burden, the time to reach 50% conversion is shortened to duereduced reserves for calcium phosphate absorption. Thus, short T50 timessuggest a reduced capacity to absorb calcium phosphate crystals whereasprolonged T50 times are consistent with high capacities. Short T50 timesare often associated with warfarin treatment, or hemodialysis or uremicoxidative load, and generally are associated with increased risk ofmyocardial infarctions, heart failure and all-cause mortality.

Without wishing to be bound by theory, it is believed that theadministration of MK-7 and/or MKH2-7 increases T50 times and improving asubject's capacity to prevent pathologic calcification. Because subjectswith CKD and ESRD exhibit reduced levels of carboxylated Matrix GlaProtein, which is Vitamin K-dependent and essential for the formation ofprimary CPP, administration of MK-7 and/or MKH2-7 in accordance with themethods of the present disclosure can reduce the risk for pathologiccalcification and prevent the development of calciphylaxis.

Accordingly, in certain embodiments of the methods disclosed herein,administration of the MK-7 and/or MKH2-7 to the subject increases thesubject's serum T50 value (e.g., by at least 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100%) relative to the subject's serum T50 value prior to administrationof the MK-7 and/or MKH2-7. Methods for measuring a subject's serum T50value are known in the art. (See, e.g., Pasch et al. (2012)“Nanoparticle-based test measures overall propensity for calcificationin serum.” J. AM. SOC. NEPHROL. 23(10): 1744-52; Dahle et al., (2016)“Serum Calcification Propensity Is a Strong and Independent Determinantof Cardiac and All-Cause Mortality in Kidney Transplant Recipients.” AM.J. TRANSPLANT 16(1): 204-12; and Smith et al. (2014) “SerumCalcification Propensity Predicts All-Cause Mortality in PredialysisCKD,” J. AM. SOC. NEPHROL. 25(2):339-348).

In certain embodiments, the disclosure relates to a method of increasinga serum T50 value in a subject having a disorder as described herein,the method comprising administering at least 2 mg/day (e.g., at least 2mg of substantially pure MK-7 and/or MKH2-7 per day, e.g., 10, 15, 25,50 or 75 mg per day) MK-7 and/or MKH2-7 to the subject, whereinadministration of the MK-7 and/or MKH2-7 to the subject increases thesubject's serum T50 value (e.g., by at least 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100%) relative to the subject's serum T50 value prior to administrationof the MK-7 and/or MKH2-7.

2. Carboxylation of Vitamin K-Dependent Proteins

Despite dietary deficiencies, Vitamin K levels among ESRD patients maynot be reduced. For example, a study of 172 stable hemodialysis patientsfound that only 6% of patients exhibited a clinically significantdeficiency in Vitamin K. (Holden et al. (2010) “Vitamins K and D Statusin Stages 3-5 Chronic Kidney Disease,” CLIN. J. AM. Soc. NEPHROL.5:590-597.) However, when patients were examined for the level ofcarboxylated osteocalcin, a Vitamin K-dependent protein, approximately60% of patients had reduced levels. (Id.) Similar results were shown forother Vitamin K-dependent proteins, such as PIVKA-II. (Id., and Pilkeyet al. (2007) “Subclinical Vitamin K Deficiency in HemodialysisPatients,” AM. J. KIDNEY DIS. 49:432-439.) These results are consistentwith the hypothesis that in uremic patients, total Vitamin K levels canbe normal while generation of reduced forms are blocked by the oxidativeproperties of uremia. (Id.)

The observation that oxidant conditions can disrupt the Vitamin K cyclesuggests that the oxidant load generated during hemodialysis furthercontributes to the high rates of soft tissue (e.g., vascular)calcification observed within the ESRD population. Delivery ofhemodialysis is known to lead to the oxidation of numerous tissueproteins, and both CKD and ESRD patients exhibit a higher percentage(15-fold) of carbonyl proteins compared to normal controls.

Without wishing to be bound by the theory, it is believed that theoxidative load generated by uremia and/or hemodialysis leads tooxidation of the functional Vitamin K hydroquinone (KH2) to thenon-functional native vitamin. The oxidation of KH2 blocks its abilityto function as a co-factor for GGCX which leads to reduced gammacarboxylation of Vitamin K-dependent proteins. Thus, administration ofMK-7 and/or MKH2-7 to subjects with diabetes, CKD, and/or ESRD and/orsubjects undergoing hemodialysis, can increase the ratio of one or morecarboxylated Vitamin K-dependent proteins to non-carboxylated VitaminK-dependent proteins in these subjects and/or decrease the amount of oneor more non-carboxylated Vitamin K-dependent proteins in these subjects.

Accordingly, in certain embodiments, administration of MK-7 and/orMKH2-7 increases a ratio of a carboxylated to a non-carboxylated VitaminK-dependent protein in the subject's plasma (e.g., by at least 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100%) relative to the ratio prior to administration ofthe MK-7 and/or MKH2-7. In certain embodiments, administration of MK-7and/or MKH2-7 increases a ratio of a carboxylated to a non-carboxylatedVitamin K-dependent protein in the subject's plasma by between about 10%and 500% or higher, between about 30% and about 500%, between about 30%and about 200%, between about 30% and about 100%. In certainembodiments, administration of MK-7 and/or MKH2-7 increases a ratio of acarboxylated to a non-carboxylated Vitamin K-dependent protein in thesubject's plasma by between about 50% and about 500%, between about 50%and about 200%, between about 50% and about 100%. In certainembodiments, administration of MK-7 and/or MKH2-7 increases a ratio of acarboxylated to a non-carboxylated Vitamin K-dependent protein in thesubject's plasma by between about 70% and about 500%, between about 70%and about 200%, between about 70% and about 100%.

In certain embodiments, administration of MK-7 and/or MKH2-7 reduces anamount of non-carboxylated Vitamin K-dependent protein in the subject'splasma (e.g., by at least 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) relative to theamount prior to administration of the MK-7 and/or MKH2-7. In certainembodiments, administration of MK-7 and/or MKH2-7 reduces an amount of anon-carboxylated Vitamin K-dependent protein in the subject's plasma bybetween about 5% and about 100%, between about 5% and about 80%, betweenabout 5% and about 30%, between about 20% and about 100%, or betweenabout 20% and about 100%.

In certain embodiments, the disclosure relates to a method forincreasing a ratio of a carboxylated to a non-carboxylated VitaminK-dependent protein in the plasma of a subject having a disorder asdescribed herein, the method comprising administering at least 2 mg/day(e.g., at least 2 mg of substantially pure MK-7 and/or MKH2-7 per day,e.g., 10, 15, 25, 50 or 75 mg per day) MK-7 and/or MKH2-7 to thesubject, wherein administration of the MK-7 and/or MKH2-7 to the subjectincreases a ratio of a carboxylated to a non-carboxylated VitaminK-dependent protein in the subject's plasma (e.g., by at least 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100%) relative to the ratio prior to administration ofthe MK-7 and/or MKH2-7.

In certain embodiments, the disclosure relates to a method fordecreasing an amount of a non-carboxylated Vitamin K-dependent proteinin the plasma of a subject having a disorder as described herein, themethod comprising administering at least 2 mg/day (e.g., at least 2 mgof substantially pure MK-7 and/or MKH2-7 per day, e.g., 10, 15, 25, 50or 75 mg per day) MK-7 and/or MKH2-7 to the subject, whereinadministration of the MK-7 and/or MKH2-7 to the subject decreases anamount of a non-carboxylated Vitamin K-dependent protein in thesubject's plasma (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%)relative to the amount prior to administration of the MK-7 and/orMKH2-7.

In certain embodiments, the Vitamin K-dependent protein is selected fromMatrix Gla Protein (MGP), Growth Arrest Specific Gene 6 (Gas-6) protein,PIVKA-II protein, osteocalcin, activated Protein C, or activated ProteinS. Carboxylated forms of MGP, Gas-6, PIVKA-II, osteocalcin, activatedProtein C and activated Protein S can be measured using sandwich assays,as is known in the art. (See, e.g., Nigwekar et al. (2017), supra(detecting MGP); Gla-Type Osteocalcin (GLa-OC) ELISA Kit (Takara,Mountain View, Calif.); STA-Staclot Protein C (Stago, Asnieres surSeine, France; and REAADS Protein S Antigen, cat. no. K036-001,Diapharma Group, West Chester, Ohio). An increase in the ratio ofcarboxylated to non-carboxylated Vitamin K-dependent proteins in asubject following administration with MK-7 is indicative of successfulrescue of the GGCX carboxylation system. Accordingly, effective MK-7and/or MKH2-7 administration can lead to an increase in the ratio ofcarboxylated to non-carboxylated proteins in the subject.

PIVKA-II is an abnormal form of prothrombin which has not beencarboxylated, also known as des-gamma carboxyprothrombin (DCP). Thus,elevated levels of PIVKA-II correspond to poorer vitamin K status. As abiomarker of vitamin K status, PIVKA-II has the advantage of beingindependent of kidney function and lipid profile (Elliot et al. (2014)“Assessment of potential biomarkers of subclinical vitamin K deficiencyin patients with end-stage kidney disease,” CAN. J. KIDNEY HEALTH DIS.1:13.) According to the methods herein, measuring PIVKA-II in a subjectis indicative of successful rescue of the GGCX carboxylation system.PIVKA-II plasma levels can be measured by any method known in the art,e.g., using ELISA (Diagnostica Stage, Parsippany, N.J.). (See also,e.g., Dituri et al. (2012) “PIVKA-II plasma levels as markers ofsubclinical vitamin K deficiency in term infants,” J. MATERNAL-FETAL &NEONATAL MEDICINE 25 (9): 1660-1663.)

MGP is believed to be a potent calcification inhibitor of the arterialwall and other soft tissues, where its activity depends on vitaminK-dependent γ-glutamate carboxylation (Schurgers et al. (2007a), supra;Westenfeld et al. (2012) “Effect of vitamin K₂ supplementation onfunctional vitamin K deficiency in hemodialysis patients: a randomizedtrial,” AM. J. KIDNEY DIS. 59(2):186-195; Schurgers et al. (2013)“Vitamin K-dependent carboxylation of matrix Gla-protein: a crucialswitch to control ectopic mineralization,” TRENDS MOL MED 2013;19(4):217-26; Marles et al. (2017) “US Pharmacopeial Convention safetyevaluation of menaquinone-7, a form of vitamin K,” NUTR. REV.75(7):553-578). Uncarboxylated MGP, formed as a result of vitamin Kdeficiency, is associated with cardiovascular disease. Recent studiessuggest poor vitamin K status in hemodialysis patients (Westenfeld etal. (2012), supra). The circulating inactive form of MGP (dp-ucMGP) wasshown to increase progressively in patients with CKD (Schurgers et al.(2010), supra) and to be predictive of vitamin K status and correlatedwith vascular calcification in patients on hemodialysis (Delanaye et al.(2014), supra). Plasma dp-ucMGP has therefore been proposed as asurrogate marker for vascular calcification in CKD (Schurgers et al.(2010), supra). In addition, lower levels of circulatingdesphospho-carboxylated MGP (dp-cMGP) was proposed as a predictor ofmortality in hemodialysis patients (Schlieper et al. (2011) “Circulatingnonphosphorylated carboxylated matrix gla protein predicts survival inESRD,” J. AM. SOC. NEPHROL. 22(2):387-95).

Osteocalcin is a non-collagenous bone matrix protein synthesized bymature osteoblasts, and involved in bone formation and re-gelation ofbone mineralization (Elliott et al. 2014, supra; Marles et al. 2017,supra). The proportion of osteocalcin that is uncarboxylated is asensitive marker of vitamin K status in bone, and subclinical vitamin Kdeficiency is defined by an increase in the proportion of uncarboxylatedosteocalcin above 20% (Elliott et al. 2014, supra). Supplementation withMK-7 at doses of 100-200 μg/d for 4 to 12 weeks was shown to increasethe ratio of carboxylated to undercarboxylated osteocalcin significantlyand in a dose-dependent manner (Inaba et al. (2015), “Low-Dose DailyIntake of Vitamin K(2) (Menaquinone-7) Improves Osteocalcinγ-Carboxylation: A Double-Blind, Randomized Controlled Trials,” J. NUTR.SCI. VITAMINOL. 61(6):471-80).

Vitamin K-dependent γ-carboxyglutamate proteins are synthesized in theliver, and help maintain normal blood coagulation through a balance ofboth procoagulant factors (II, VII, IX, and X) and anticoagulantproteins (C and S) (Schurgers et al. (2007c) “Vitamin K-containingdietary supplements: comparison of synthetic vitamin K₁ andnatto-derived menaquinone-7,” BLOOD 109(8):3279-83; Marles et al. (2017)supra). Protein C regulates the coagulation process by neutralizing theprocoagulant activities of factors V and VIII in the presence of thecofactor Protein S (Marlar et al. (2017) “Assessment of HereditaryThrombophilia: Performance of Protein C (PC) Testing,” METHODS MOL.BIOL. 1646:145-151). Hypercoagulable states, such as protein C and/orprotein S deficiencies, have been reported in patients withcalciphylaxis, and proposed as factors increasing the likelihood ofcalciphylaxis development (Wilmer et al. (2002), supra; Nigwekar et al.(2008) “Calciphylaxis from nonuremic causes: a systematic review,” CLIN.J. AM. SOC. NEPHROL. 3(4):1139-43). In a randomized study evaluating theeffect of vitamin K₂ supplementation on functional vitamin K deficiencyin adult hemodialysis patients, patients on hemolysis (N=53) had4.5-fold higher dp-ucMGP and 8.4-fold higher uncarboxylated osteocalcinlevels compared with healthy age-matched controls (N=50). PIVKA-IIlevels were elevated in 49 hemodialysis patients. Vitamin K2supplementation induced a dose- and time-dependent decrease incirculating dp-ucMGP, uncarboxylated osteocalcin, and PIVKA-II levels.Response rates in the reduction in dp-ucMGP levels were 77% and 93% inthe groups receiving 135 μg and 360 μg of menaquinone-7, respectively(Westenfeld et al. (2012) supra).

3. Levels of Other Biomarkers for Calcification

In certain embodiments, administration of the MK-7 and/or MKH2-7increases the plasma level of osteoprotegerin or Fetuin A (e.g., by atleast 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 100%) relative to the plasmaconcentration of osteoprotegerin or Fetuin A prior to administration ofthe MK-7 and/or MKH2-7. In certain embodiments, administration of theMK-7 and/or MKH2-7 increases the plasma level of osteoprotegerin orFetuin A by about 10-50%, by about 50-100%, by about 100-200%, or byabout 200-500% relative to the plasma concentration of osteoprotegerinor Fetuin A prior to administration of the MK-7 and/or MKH2-7.Osteoprotegerin and Fetuin A are inhibitors of tissue calcification.Accordingly, an increase in levels of osteoprotegerin and/or Fetuin A isindicative that administration of the MK-7 and/or MKH2-7 is successfullypreventing, slowing the progression of, arresting, and/or reversingcalciphylaxis.

In certain embodiments, the disclosure relates to a method forincreasing the plasma level of osteoprotegerin or Fetuin A in a subjecthaving a disorder as described herein, the method includingadministering at least 2 mg/day (e.g., at least 2 mg of substantiallypure MK-7 and/or MKH2-7 per day, e.g., 10, 15, 25, 50 or 75 mg per day)MK-7 and/or MKH2-7 to the subject, wherein administration of the MK-7and/or MKH2-7 to the subject increases the plasma level ofosteoprotegerin or Fetuin A (e.g., by at least 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100%) relative to the plasma concentration of osteoprotegerin or FetuinA prior to administration of the MK-7 and/or MKH2-7.

In certain embodiments of any of the above aspects, administration ofthe MK-7 and/or MKH2-7 decreases the plasma level of D-Dimers (e.g., byat least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 100%) relative to the plasmaconcentration of D-Dimers prior to administration of the MK-7 and/orMKH2-7. In certain embodiments of any of the above aspects,administration of the MK-7 and/or MKH2-7 decreases the plasma level ofD-Dimers by about 10-50%, by about 50-100%, or by about 10-100% relativeto the plasma concentration of D-Dimers prior to administration of theMK-7 and/or MKH2-7. D-Dimers are indicative of the functional status ofVitamin D metabolism. Protein C and Protein S prevent generation ofthrombin and fibrin, and the lack of functional Vitamin K contributes toa loss of Protein C and Protein S, which can lead to microthrombosis viadegradation of fibrin and formation of D-Dimers. Thus presence ofD-Dimers is an indicator of sub-clinical thrombosis. Accordingly,reduction in the plasma level of D-Dimer may indicate thatadministration of the MK-7 and/or MKH2-7 is successfully restoring theGGCX carboxylation system, which can lead to preventing, slowing theprogression of, arresting, and/or reversing calciphylaxis.

In certain embodiments, the disclosure relates to a method fordecreasing the plasma level of D-Dimers in a subject having a disorderas described herein, the method including administering at least 2mg/day (e.g., at least 2 mg of substantially pure MK-7 and/or MKH2-7 perday, e.g., 10, 15, 25, 50 or 75 mg per day) MK-7 and/or MKH2-7 to thesubject, wherein administration of the MK-7 and/or MKH2-7 to the subjectdecreases the plasma level of D-Dimers (e.g., by at least 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100%) relative to the plasma concentration of D-Dimersprior to administration of the MK-7 and/or MKH2-7.

In certain embodiments of any of the above aspects, administration ofthe MK-7 and/or MKH2-7 decreases the plasma level of Highly Sensitive CReactive Protein (hs-CRP) (e.g., by at least 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100%) relative to the plasma concentration of Highly Sensitive CReactive Protein (hs-CRP) prior to administration of the MK-7 and/orMKH2-7. In certain embodiments of any of the above aspects,administration of the MK-7 and/or MKH2-7 decreases the plasma level ofHighly Sensitive C Reactive Protein (hs-CRP) by about 10-50%, by about50-100%, or by about 10-100% relative to the plasma concentration ofHighly Sensitive C Reactive Protein (hs-CRP) prior to administration ofthe MK-7 and/or MKH2-7. Hs-CRP is a biomarker for ongoing inflammation.The presence of inflammation enhances calciphylaxis. Accordingly,reduction in the plasma level of hs-CRP can be indicative thatadministration of the MK-7 and/or MKH2-7 is successfully preventing,slowing the progression of, arresting, and/or reversing calciphylaxis.

In certain embodiments, the disclosure relates to a method fordecreasing the plasma level of Highly Sensitive C Reactive Protein(hs-CRP) in a subject having a disorder as described herein, the methodincluding administering at least 2 mg/day (e.g., at least 2 mg ofsubstantially pure MK-7 and/or MKH2-7 per day, e.g., 10, 15, 25, 50 or75 mg per day) MK-7 and/or MKH2-7 to the subject, wherein administrationof the MK-7 and/or MKH2-7 to the subject decreases the plasma level ofHighly Sensitive C Reactive Protein (hs-CRP) (e.g., by at least 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100%) relative to the plasma concentration of HighlySensitive C Reactive Protein (hs-CRP) prior to administration of theMK-7 and/or MKH2-7.

V. Lesions

The methods and compositions described herein can be useful in arrestingor stopping the progression of, or reversing tissue lesions, for exampledermal lesions or vascular lesions.

1. Dermal Lesions

The final step of the Vitamin K cycle requires the enzymatic reductionof Vitamin K 2,3-epoxide back to MK-7 by Vitamin K epoxide reductasecomplex subunit 1 (VKORC1, also referred to as VKOR (see, FIG. 1). Insome tissues, the paralog VKORC1L1 (VKORC1-Like-1) may also perform thereaction. Skin exhibits the lowest expression of VKOR-C1, consistentwith the clinical observation that Vitamin K-dependent vascularcalcification is more common in the dermis. Low skin expression ofVKOR-C1 also suggests that any condition or procedure that blocks thegeneration of Vitamin K, such as hemodialysis, predisposes skin topathologic calcification.

Thus, the methods and compositions described herein relate in part toreducing the size of a dermal lesion. In certain embodiments, thesubject has a dermal lesion, and the administration of the MK-7 and/orMKH2-7 reduces the size of the dermal lesion. In certain embodiments,administration of the MK-7 and/or MKH2-7 reduces the total surface areaof the dermal lesion by at least 55%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45% 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. Incertain embodiments, administration of the MK-7 and/or MKH2-7 reducesthe total volume of the dermal lesion by at least 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 100% (i.e., the dermal lesion is eliminated).

In certain embodiments, administration of MK-7 and/or MKH2-7 reduces thetotal surface area or volume of the dermal lesion by between about 30%and about 100%, between about 30% and about 90%, between about 30% andabout 80%, between about 30% and about 70%, or between about 30% andabout 60%. In certain embodiments, administration of MK-7 and/or MKH2-7reduces the total surface area or volume of the dermal lesion by betweenabout 50% and about 100%, between about 50% and about 90%, between about50% and about 80%, between about 50% and about 70%, or between about 50%and about 60%. In certain embodiments, administration of MK-7 and/orMKH2-7 reduces the total surface area or volume of the dermal lesion bybetween about 70% and about 100%, between about 70% and about 90%,between about 70% and about 80%.

The surface area or volume of one or more skin lesions can be measuredby any method known in the art. When surface area is measured, a meantotal surface area can be calculated if multiple lesions are present.Volume of a lesion can be calculated from a three-dimensional model ofthe lesion which is constructed from digital imaging.

Further, prior to the development of outward signs of a dermal lesion,the size of the dermal lesion can be measured by digital analysis. Inanother embodiment, the effectiveness of administration of MK-7 and/orMKH2-7 is determined by Von Kossa staining for calcium in a dermalbiopsy. This well-established stain is a validated means to examine andquantify the amount of interstitial calcium deposition in a dermalbiopsy. In this approach, a skin biopsy is taken and the tissue isstained using the Von Kossa method. By binding to soft tissue andvascular deposits of phosphate, Von Kossa staining can be used todetermine whether the rate of soft tissue and vascular calcium phosphatedeposition is being slowed or reversed with administration of MK-7and/or MKH2-7. Because the calcium phosphate deposits are central topathogenesis of calciphylaxis, a reduction in Von Kossa stainingindicates a healing of the conditions that lead to calciphylaxislesions. Kits for performing the Von Kossa method are availablecommercially (see, e.g., Abeam®, # ab150687).

In certain embodiments, administration of the MK-7 and/or MKH2-7 reducesthe dermal lesion to 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%,45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% (i.e., the dermallesion is eliminated) of the total surface of the lesion prior totreatment.

2. Vascular Lesions

The methods and compositions described herein relate in part to reducinga vascular lesion. In certain embodiments, the subject has a vascularlesion, and the administration of the MK-7 and/or MKH2-7 reduces thevascular lesion. Reduction in a vascular lesion can be assessed usingaortic plethysmography, which measures vascular compliance (i.e., thecapacity of a blood vessel to dilate). (See, e.g., Inuma et al. (2012)HONG KONG JOURNAL OF NEPHROLOGY 14(2):48-53.)

Reduction in a vascular lesion also can be assessed by measuring pulsewave velocity (PWV). PWV is the velocity at which the arterial pulsemoves through the circulatory system and is used as a measure ofarterial stiffness. Higher PWV corresponds to higher arterial stiffness,and therefore lower vascular compliance. Methods for measuring PWV areknown in the art and described, for example, by Pereira et al. (2015),supra. Briefly, PWV can be assessed by measuring regional PWV, such ascarotid-femoral PWV, or local PWV. Methods for measuring regional PWVare known in the art and include, for example, the use of a pulsetransducer probe, TY-360 pressure transducer, doppler unit synchronismwith electrocardiogram (ECG), pulse transducer probes,photoplethysmography, Complior® (piezoelectric pressure transducers),SphygmoCor®, Arteriograph®, and PulsePen® (tonometer and integratedelectrocardiogram unit). Id. Methods for measuring local PWV are knownin the art and include, for example, magnetic resonance imaging,ultrasound, and angiography. Id.

In certain embodiments, administration of the MK-7 and/or MKH2-7increases vascular compliance of a blood vessel by at least 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100% relative to vascular compliance prior totreatment. In certain embodiments, administration of the MK-7 and/orMKH2-7 increases vascular compliance of a blood vessel by about 10-50%,by about 50-100%, by about 100-200%, or by about 200-500%. In certainembodiments, administration of the MK-7 and/or MKH2-7 increases vascularcompliance of a blood vessel by at least 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%relative to vascular compliance prior to treatment. In certainembodiments, administration of the MK-7 and/or MKH2-7 increases vascularcompliance of a blood vessel by about 10-50%, by about 50-100%, by about100-200%, or by about 200-500% relative to vascular compliance prior totreatment.

VI. Kits for Use in Medical Applications

Another aspect of the invention provides a kit for treating one or moreof the disorders or conditions described herein. The kit can include: i)instructions for treating the medical disorder; and ii) one or moredosage units containing substantially pure MK-7 and/or MKH2-7.

Throughout the description, where compositions are described as having,including, or comprising specific components, or where processes andmethods are described as having, including, or comprising specificsteps, it is contemplated that, additionally, there are compositions ofthe present invention that consist essentially of, or consist of, therecited components, and that there are processes and methods accordingto the present invention that consist essentially of, or consist of, therecited processing steps.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

Throughout the description, where compositions and kits are described ashaving, including, or comprising specific components, or where processesand methods are described as having, including, or comprising specificsteps, it is contemplated that, additionally, there are compositions andkits of the present invention that consist essentially of, or consistof, the recited components, and that there are processes and methodsaccording to the present invention that consist essentially of, orconsist of, the recited processing steps.

In the application, where an element or component is said to be includedin and/or selected from a list of recited elements or components, itshould be understood that the element or component can be any one of therecited elements or components, or the element or component can beselected from a group consisting of two or more of the recited elementsor components.

Further, it should be understood that elements and/or features of acomposition or a method described herein can be combined in a variety ofways without departing from the spirit and scope of the presentinvention, whether explicit or implicit herein. For example, wherereference is made to a particular compound, that compound can be used invarious embodiments of compositions of the present invention and/or inmethods of the present invention, unless otherwise understood from thecontext. In other words, within this application, embodiments have beendescribed and depicted in a way that enables a clear and conciseapplication to be written and drawn, but it is intended and will beappreciated that embodiments may be variously combined or separatedwithout parting from the present teachings and invention(s). Forexample, it will be appreciated that all features described and depictedherein can be applicable to all aspects of the invention(s) describedand depicted herein.

The articles “a” and “an” are used in this disclosure to refer to one ormore than one (i.e., to at least one) of the grammatical object of thearticle, unless the context is inappropriate. By way of example, “anelement” means one element or more than one element.

The term “and/or” is used in this disclosure to mean either “and” or“or” unless indicated otherwise.

It should be understood that the expression “at least one of” includesindividually each of the recited objects after the expression and thevarious combinations of two or more of the recited objects unlessotherwise understood from the context and use. The expression “and/or”in connection with three or more recited objects should be understood tohave the same meaning unless otherwise understood from the context.

The use of the term “include,” “includes,” “including,” “have,” “has,”“having,” “contain,” “contains,” or “containing,” including grammaticalequivalents thereof, should be understood generally as open-ended andnon-limiting, for example, not excluding additional unrecited elementsor steps, unless otherwise specifically stated or understood from thecontext.

Where the use of the term “about” is before a quantitative value, thepresent invention also include the specific quantitative value itself,unless specifically stated otherwise. As used herein, the term “about”refers to a ±10% variation from the nominal value unless otherwiseindicated or inferred.

Where a molecular weight is provided and not an absolute value, forexample, of a polymer, then the molecular weight should be understood tobe an average molecule weight, unless otherwise stated or understoodfrom the context.

As a general matter, compositions specifying a percentage are by weightunless otherwise specified. Further, if a variable is not accompanied bya definition, then the previous definition of the variable controls.

It should be understood that the order of steps or order for performingcertain actions is immaterial so long as the present invention remainoperable. Moreover, two or more steps or actions may be conductedsimultaneously.

The use of any and all examples, or exemplary language herein, forexample, “such as” or “including,” is intended merely to illustratebetter the present invention and does not pose a limitation on the scopeof the invention unless claimed. No language in the specification shouldbe construed as indicating any non-claimed element as essential to thepractice of the present invention.

EXAMPLES

The disclosure is further illustrated by the following examples, whichare not to be construed as limiting this disclosure in scope or spiritto the specific procedures herein described. It is to be understood thatthe examples are provided to illustrate certain embodiments and that nolimitation to the scope of the disclosure is intended thereby.

Example 1—Administration of MK-7 and/or MKH2-7 in Subjects at Risk forDevelopment of Calciphylaxis

This example describes the administration of MK-7 and/or MKH2-7 tosubjects at risk for development of calciphylaxis, but who have not yetdeveloped the characteristic skin lesions of calciphylaxis. Risk factorsto be considered include, but are not limited to, diabetes mellitus,obesity, ESRD and/or hemodialysis, and prior treatment with warfarin(Nigwekar et al. (2016), supra). It is contemplated that administrationof MK-7 and/or MKH2-7 can result in protection of the subjects from skinlesions and a change in certain biomarker levels indicative of theprevention of the development of calciphylaxis.

Subjects at risk of development of calciphylaxis orally receive MK-7and/or MKH2-7 at 10 mg, 25 mg or 50 mg once daily for at least 2 weeks,4 weeks, 6 weeks, 8 weeks, 3 months, 6 months, 1 year, or indefinitely.The dosage form is a 10 mg or 25 mg soft-gel capsule. Two 25 mg capsulesare be administered once daily to the 50 mg dosage cohort.

It should be noted that not all subjects with elevated risk factors forcalciphylaxis will develop the characteristic skin lesions ofcalciphylaxis. The intent of treating with MK-7 and/or MKH2-7proactively (prior to a clinical diagnosis of calciphylaxis) is theprevention of lesion appearance. Thus, a drop in frequency of, orelimination of lesion appearances is contemplated to be a successfultreatment.

Several biomarkers can be assessed to determine the efficacy of MK-7and/or MKH2-7 at the three dose levels. Exemplary biomarkers includePIVKA-II; uncarboxylated and total Matrix Gla Protein (MGP);uncarboxylated, carboxylated and total osteocalcin protein;uncarboxylated, carboxylated and total Protein C, osteoprotegerin,Fetuin A and hs-CRP.

Blood samples are obtained to measure the biomarkers according to thefollowing schedule. Blood sampling can occur during treatment on aweekly or monthly basis. It is contemplated that administration of MK-7and/or MKH2-7 will result in (i) an increase in PIVKA-II,osteoprotegerin, or Fetuin A, which is indicative of slowing theprogression of, arresting, or reversing, calciphylaxis, (ii) a decreasein uncarboxylated MGP, uncarboxylated osteocalcin, and/or uncarboxylatedProtein C, which is indicative of slowing the progression of, arresting,or reversing calciphylaxis.

Further, pulse wave velocity (PWV) can be measured to assess arterialcompliance. Improved vascular compliance will be indicative of slowingthe progression of, arresting, or reversing calciphylaxis.

Example 2—Administration of MK-7 and/or MKH12-7 in Subjects at Diagnosedwith Calciphylaxis

This example describes the administration of MK-7 and/or MKH2-7 tosubjects diagnosed with calciphylaxis. Typical symptoms includepresentation of characteristic painful skin lesions (Nigwekar et al.(2015) “Calciphylaxis: Risk Factors, Diagnosis, and Treatment.” Am. J.Kidney Dis. 66:133-46). Definitive diagnosis of calciphylaxis isachieved via skin biopsy (Nigwekar, 2015 supra). Further conditions needto be considered for correct diagnosis (Id.).

Subjects diagnosed with calciphylaxis orally receive MK-7 and/or MKH2-7at 10 mg, 25 mg or 50 mg once daily for at least 2 weeks, 4 weeks, 6weeks, 8 weeks, 3 months, 6 months, 1 year, or indefinitely. The dosageform is a 10 mg or 25 mg soft-gel capsule. Two 25 mg capsules areadministered once daily to the 50 mg dosage cohort.

The arrest of, or decreases in lesion size and frequency is contemplatedto be an indication of successful treatment. It is contemplated thatadministration of MK-7 and/or MKH2-7 according to the foregoing willresult in the arrest of, or decrease in lesion size and frequency.

Additionally, because calciphylaxis has a considerable mortality risk,increased overall survival time of diagnosed subjects will be anindication of treatment success. It is contemplated that administrationof MK-7 and/or MKH2-7 according to the foregoing will result in anincreased overall survival time of diagnosed subjects.

Example 3—Administration of MK-7 and/or MKH2-7 in Subjects with StableEnd Stage Renal Disease (ESRD) Receiving Hemodialysis

This example describes the administration of MK-7 to subjects with ESRDreceiving hemodialysis, and who are at risk of developing calciphylaxis.Administration of MK-7 can result in a change in certain biomarkerlevels indicative of the prevention of the development of calciphylaxis,or in the arrest or slowing down of the development of calciphylaxis.

A number of subjects with stable ESRD but without calciphylaxis areenrolled in the study, whereupon the subjects orally receive a 10 mgcapsule of MK-7 per day for 14 days. The levels of certain biomarkers,including uncarboxylated Matrix Gla Protein (MGP), uncarboxylatedosteocalcin, osteoprotegerin, Fetuin A and hs-CRP were assessed on day 1and on day 15 (the end of treatment), and the changes between day 1 andday 15 were calculated. Between days 1 and 15, the average reduction inuncarboxylated MGP was −21.9% and the average reduction inuncarboxylated osteocalcin was −55.5%. In addition, subjectsunexpectedly exhibited an average increase in osteoprotegerin and FetuinA and a reduction in hs-CRP. Osteoprotegerin, Fetuin A, and hs-CRP arenot Vitamin-K dependent proteins and it is believed that changes inthese proteins have not previously been demonstrated as a result of MK-7intake. The average increase in osteoprotegerin was 14.5%, the averageincrease in Fetuin A was 23.1%, and the average reduction in hs-CRP was−35.8%.

Based on the study, the administration of MK-7 can result in a decreasein uncarboxylated MGP, uncarboxylated osteocalcin, and hs-CRP, and anincrease in Fetuin A and osteoprotegerin, which can be indicative thatadministration of MK-7 prevents, or stops or slows down the progressionof calciphylaxis in the subjects.

INCORPORATION BY REFERENCE

The entire disclosure of each of the patent and scientific documentsreferred to herein is incorporated by reference for all purposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

What is claimed is:
 1. A method of preventing, slowing the progressionof, arresting, and/or reversing calciphylaxis in a human subject in needthereof, the method comprising administering to the human subject atleast 10 mg of substantially pure menaquinone-7 (MK-7) and/ormenaquinol-7 (MKH2-7) per day thereby to prevent, slow the progressionof, arrest, or reverse calciphylaxis in the subject, whereupon theadministration of the MK-7 and/or MKH2-7 increases a plasma level ofFetuin A relative to the plasma level of Fetuin A prior toadministration.
 2. The method of claim 1, wherein the subject has distalcalciphylaxis and/or central calciphylaxis.
 3. The method of claim 1,wherein the subject has diabetes, chronic kidney disease or end stagerenal disease.
 4. The method of claim 3, wherein the subject has stage 3chronic kidney disease.
 5. The method of claim 3, wherein the subjecthas stage 4 chronic kidney disease.
 6. The method of claim 3, whereinthe subject has stage 5 chronic kidney disease.
 7. The method of claim1, wherein the subject is undergoing hemodialysis.
 8. The method ofclaim 1, wherein the subject is receiving non-warfarin-basedanti-coagulant therapy.
 9. The method of claim 8, wherein theanti-coagulant therapy is oral anti-coagulation therapy.
 10. The methodof claim 8, wherein the anti-coagulation therapy comprises an inhibitorof Factor Xa activity or Factor IIa activity.
 11. The method of claim 1,wherein the subject has chronic obstructive pulmonary disease (COPD).12. The method of claim 1, wherein the subject has acalciphylaxis-related dermal lesion.
 13. The method of claim 12, whereinadministration of the composition reduces the size of the dermal lesion.14. The method of claim 1, whereupon administration of the MK-7 and/orMKH2-7 to the subject increases the subject's serum T50 value relativeto the subject's serum T50 value prior to administration of the MK-7and/or MKH2-7.
 15. The method of claim 1, wherein administration of theMK-7 and/or MKH2-7 (a) increases a ratio of a carboxylated to anon-carboxylated of a Vitamin K dependent protein or (b) decreases anamount of a non-carboxylated Vitamin K dependent protein in plasma ofthe subject relative to the ratio or amount present prior toadministration of the MK-7 and/or MKH2-7.
 16. The method of claim 15,wherein the Vitamin K-dependent protein is selected from Matrix GlaProtein, Growth Arrest Specific Gene 6 (Gas-6) protein, PIVKA-IIprotein, osteocalcin, activated Protein C, or activated Protein S. 17.The method of claim 1, wherein upon administration of the MK-7 and/orMKH2-7 to the subject increases a plasma level of osteoprotegerinrelative to the plasma level of osteoprotegerin prior to administrationof the MK-7 and/or MKH2-7.
 18. The method of claim 1, wherein uponadministration of the MK-7 and/or MKH2-7 to the subject decreases aplasma level of D-Dimer or Highly Sensitive C Reactive Protein (hs-CRP)relative to the plasma level of D-Dimer or Highly Sensitive C ReactiveProtein (hs-CRP) prior to administration of the MK-7 and/or MKH2-7. 19.The method of claim 1, wherein from about 10 mg to about 50 mg of MK-7and/or MKH2-7 is administered to the subject per day.
 20. The method ofclaim 1, wherein 10, 25, 50, 75 or 100 mg of MK-7 and/or MKH2-7 isadministered to the subject per day.
 21. The method of claim 1, whereinthe composition is administered to the subject for at least 2 weeks. 22.The method of claim 1, wherein the composition is administered daily.23. The method of claim 1, wherein the composition is administeredorally.
 24. The method of claim 1, wherein the subject is takingcalcium-based phosphate binders and/or vitamin D analogs.
 25. The methodof claim 1, wherein the composition is administered to the subject forat least 6 weeks.
 26. The method of claim 7, wherein the composition isadministered to the subject for a period that includes the duration ofhemodialysis.
 27. The method of claim 1, wherein the composition isdisposed within a tablet, caplet, or capsule.
 28. The method of claim 1,wherein the composition comprises MK-7 and/or MKH2-7 and apharmaceutically acceptable excipient.
 29. The method of claim 1,wherein, prior to administration of the composition, the subject has auremic oxidative blockade.
 30. The method of claim 1, wherein thecomposition is administered to the subject indefinitely.